The long-term objective of this research program is to develop novel therapeutic strategies targeting the bidirectional interactions between tumor cells and the stromal cells that are found in tumors. The central hypothesis of this proposal is that inhibition of fibroblast activation protein (FAP) proteolytic activity will abrogate the invasive and metastatic capabilities of tumors, leading to attenuated tumor growth and diminished metastatic potential. This hypothesis is based on the highly selective expression of human FAP by tumor stromal fibroblasts in epithelial carcinomas, but not by epithelial carcinoma cells, normal fibroblasts, or other normal tissue. Furthermore, we have shown that FAP overexpression potentiates tumor growth, and tumor growth is attenuated by polyclonal FAP inhibitory antibodies. Pathways by which FAP-expressing tumor fibroblasts participate in tumor growth can thus be therapeutically targeted for disruption by monoclonal antibodies that can be developed for clinical use. This proposal aims to characterize the binding properties of monoclonal antibodies that have been generated which target murine FAP. This has been initiated by immunizing mice with murine FAP, and screening of hybridoma supernatants by ELISA for FAP binding. Antibodies that target FAP and may inhibit FAP enzymatic activity have been identified, and will be further characterized for FAP selectivity by surface plasmon resonance, live cell binding assays, and immunohistochemistry studies. FAP antibodies will also be characterized for inhibition of FAP proteolytic activity by dipeptidyl peptidase assays, enzyme immunocapture assays, and quantitative zymography. FAP inhibitory antibodies will be administered in a colorectal cancer animal model to test the hypothesis that inhibition of FAP proteolytic activity inhibits tumor growth and metastases in vivo. Disruption of murine FAP proteolytic pathways by inhibitory antibodies resulting in inhibition of tumor growth and metastases will be considered sufficiently significant to warrant the clinical development of a novel therapeutic strategy targeting human cancers that induce FAP proteolytic activity.